The present invention relates to novel variants of the fluorescent protein GFP having improved fluorescence properties.
The discovery that Green Fluorescent Protein (GFP) from the jellyfish A. victoria retains its fluorescent properties when expressed in heterologous cells has provided biological research with a new, unique and powerful tool (Chalfie et al (1994). Science 263:802; Prasher (1995) Trends in Genetics 11:320; WO 95/07463).
Furthermore, the discovery of a blue fluorescent variant of GFP (Heim et al. (1994). Proc.Natl.Acad.Sci. 91:12501) has greatly increased the potential applications of using fluorescent recombinant probes to monitor cellular events or functions, since the availability of probes having different excitation and emission spectra permits simultaneous monitoring of more than one process.
However, the blue fluorescing variant described by Heim et al, Y66H-GFP, suffers from certain limitations: The blue fluorescence is weak (emission maximum at 448 nm), thus making detection difficult, and necessitating prolonged excitation of cells expressing Y66H-GFP. Moreover, the prolonged period of excitation is damaging to cells especially because the excitation wavelength is in the UV range, 360 nm-390 nm.
A very important aspect of using recombinant, fluorescent proteins in studying cellular functions is the non-invasive nature of the assay. This allows detection of cellular events in intact, living cells. A limitation with current fluorescent proteins is, however, that relatively high intensity light sources are needed for visualization. Especially with the blue variant, Y66H-GFP, it is necessary to excite with intensities that are damaging to most cells. It is worth mentioning that some cellular events like oscillations in intracellular signalling systems, e.g. cytosolic free calcium, are very photo sensitive. A further consequence of the low light emittance is that only high levels of expression can be detected. Obtaining such high level expression may stress the transcriptional and/or translational machinery of the cells.
The excitation spectrum of the green fluorescent protein from Aequorea Victoria shows two peaks: A major peak at 396 nm, which is in the potentially cell damaging UV range, and a lesser peak at 475 nm, which is in an excitation range that is much less harmful to cells. Heim et al.(1995), Nature, Vol. 373, p. 663-4, discloses a Ser65Thr mutation of GFP (S65T) having longer wavelengths of excitation and emission, 490 nm and 510 nm, respectively, than the wild-type GFP and wherein the fluorophore formation proceeded about fourfold more rapidly than in the wild-type GFP.
Expression of GFP or its fluorescent variants in living cells provides a valuable tool for studying cellular events and it is well known that many cells, including mammalian cells, are incubated at approximately 37xc2x0 C. in order to secure optimal and/or physiologically relevant growth. Cell lines originating from different organisms or tissues may have different relevant temperatures ranging from about 35xc2x0 C. for fibroblasts to about 38xc2x0 C.-39xc2x0 C. for mouse xcex2-cells. Experience has shown, however, that the fluorescent signal from cells expressing GFP is weak or absent when said cells are incubated at temperatures above room temperature, cf. Webb, C. D. et al., Journal of Bacteriology, October 1995, p. 5906-5911. Ogawa H. et al., Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 11899-11903, December 1995, and Lim et al. J. Biochem. 118, 13-17 (1995). The improved fluorescent variant S65T described by Heim et al. (1995) supra also displays very low fluorescence when incubated under normal culture conditions (37xc2x0 C.), cf. Kaether and Gerdes FEBS Letters 369 (1995) pp. 267-271. Many experiments involving the study of cell metabolism are dependent on the possibility of incubating the cells at physiologically relevant temperatures, i.e. temperatures at about 37xc2x0 C.
The purpose of the present invention is to provide novel fluorescent proteins, such as F64L-GFP (SEQ ID NO: 18, hereinafter referred to as F64L-GFP), F64L-Y66H-GPP (SEQ ID NO: 16, hereinafter referred to as F64L-Y66H-GFP) and F64L-S65T-GFP (SEQ ED NO: 20, hereinafter referred to as F64L-S65T-GFP) that result in a cellular fluorescence far exceeding the cellular fluorescence from cells expressing the parent proteins, i.e. GFP (SEQ ID NO: 22, hereinafter referred to as GFP), the blue variant Y66H-GFP and the S65T-GFP variant, respectively. This greatly improves the usefulness of fluorescent proteins in studying cellular functions in living cells.
A further purpose of the invention is to provide novel fluorescent proteins that exhibit high fluorescence in cells expressing them when said cells are incubated at a temperature of 30xc2x0 C. or above, preferably at a temperature of from 32xc2x0 C. to 39xc2x0 C., more preferably at a temperature of from 35xc2x0 C. to 38xc2x0 C., and most preferably at a temperature of about 37xc2x0 C.
It is known that fluorescence in wild-type GFP is due to the presence of a chromophore, which is generated by cyclisation and oxidation of the SYG at position 65-67 in the predicted primary amino acid sequence and presumably by the same reasoning of the SHG sequence and other GFP analogues at positions 65-67, cf. Heim et al. (1994). Surprisingly, we have found that a mutation, preferably a substitution, of the F amino acid residue at position 1 preceding the S of the SYG or SHG chromophore or the T of the THG chromophore, in casu position 64 in the predicted primary amino acid sequence, results in a substantial increase of fluorescence intensity apparently without shifting the excitation and emission wavelengths. This increase is remarkable for the blue variant Y66H-GFP, which hitherto has not been useful in biological systems because of its weak fluorescence.
The F64L, F64I, F64V, F64A, and F64G substitutions are preferred, the F64L substitution being most preferred, but other mutations, e.g. deletions, insertions, or posttranslational modifications immediately preceding the chromophore are also included in the invention, provided that they result in improved fluorescence properties of the various fluorescent proteins. It should be noted that extensive deletions may result in loss of the fluorescent properties of GFP. It has been shown, that only one residue can be sacrificed from the amino terminus and less than 10 or 15 from the carboxyl terminus before fluorescence is lost, cf. Cubitt et al. TIBS Vol. 20 (11), pp. 448-456, November 1995.
Accordingly, one aspect of the present invention relates to a fluorescent protein derived from Aequorea Green Fluorescent Protein (GFP) or any functional analogue thereof, wherein the amino acid in position 1 upstream from the chromophore has been mutated to provide an increase of fluorescence intensity when the fluorescent protein of the invention is expressed in cells. Surprisingly, said mutation also results in a significant increase of the intensity of the fluorescent signal from cells expressing the mutated GFP and incubated at 30xc2x0 C. or above 30xc2x0 C., preferably at about 37xc2x0 C., compared to the prior art GFP variants.
There are several advantages of the proteins of the invention, including:
Excitation with low energy light sources. Due to the high degree of brightness of F64L-Y66H-GFP and F64L-GFP their emitted light can be detected even after excitation with low energy light sources. Thereby it is possible to study cellular phenomena, such as oscillations in intracellular signalling systems, that are sensitive to light induced damage. As the intensity of the emitted light from the novel blue and green emitting fluorescent proteins are of the same magnitude, it is possible to visualize them simultaneously using the same light source.
A real time reporter for gene expression in living cells is now possible, since the fluorescence from F64L-Y66H-GFP and F64L-GFP reaches a detectable level much faster than from wild type GFP, and prior known derivatives thereof. Hence, it is more suitable for real time studies of gene expression in living cells. Detectable fluorescence may be obtained faster due to shorter maturation time of the chromophore, higher emission intensity, or a more stable protein or a combination thereof.
Simultaneous expression of the novel fluorescent proteins under control of two or more separate promoters.
Expression of more than one gene can be monitored simultaneously without any damage to living cells.
Simultaneous expression of the novel proteins using one reporter as internal reference and the other as variable marker, since regulated expression of a gene can be monitored quantitatively by fusion of a promoter to e.g. F64L-GFP (or F64L-Y66H-GFP), measuring the fluorescence, and normalizing it to the fluorescence of constitutively expressed F64L-Y66H-GFP (or F64L-GFP). The constitutively expressed F64L-Y66H-GFP (or F64L-GFP) works as internal reference.
Use as a protein tag in living and fixed cells. Due to the strong fluorescence the novel proteins are suitable tags for proteins present at low concentrations. Since no substrate is needed and visualisation of the cells do not damage the cells dynamic analysis can be performed.
Use as an organelle tag. More than one organelle can be tagged and visualised simultaneously in living cells, e.g. the endoplasmic reticulum and the cytoskeleton.
Use as markers in cell or organelle fusions. By labelling two or more cells or organelles with the novel proteins, e.g. F64L-Y66H-GFP and F64L-GFP, respectively, fusions, such as heterokaryon formation, can be monitored.
Translocation of proteins fused to the novel proteins of the invention can be visualised. The translocation of intracellular proteins to a specific organelle, can be visualised by fusing the protein of interest to one fluorescent protein, e.g. F64L-Y66H-GFP, and labelling the organelle with another fluorescent protein, e.g. F64L-GFP, which emits light of a different wavelength. Translocation can then be detected as a spectral shift of the fluorescent proteins in the specific organelle.
Use as a secretion marker. By fusion of the novel proteins to a signal peptide or a peptide to be secreted, secretion may be followed on-line in living cells. A precondition for that is that the maturation of a detectable number of novel fluorescent protein molecules occurs faster than the secretion. This appears not to be the case for the fluorescent proteins GFP or Y66H-GFP of the prior art.
Use as genetic reporter or protein tag in transgenic animals. Due to the strong fluorescence of the novel proteins, they are suitable as tags for proteins and gene expression, since the signal to noise ratio is significantly improved over the prior art proteins, such as wild-type GFP.
Use as a cell or organelle integrity marker. By co-expressing two of the novel proteins, the one targeted to an organelle and the other expressed in the cytosol, it is possible to calculate the relative leakage of the cytosolic protein and use that as a measure of cell integrety.
Use as a marker for changes in cell morphology. Expression of the novel proteins in cells allows easy detection of changes in cell morphology, e.g. blebbing, caused by cytotoxic agents or apoptosis. Such morphological changes are difficult to visualize in intact cells without the use of fluorescent probes.
Use as a transfection marker, and as a marker to be used in combination with FACS sorting. Due to the increased brightness of the novel proteins the quality of cell detection and sorting can be significantly improved.
Use of the novel proteins as a ratio real-time kinase probe. By simultaneous expression of, e.g. F64L-GFP (or F64L-Y66H-GFP), which emits more light upon phophorylation and a derivative of F64L-Y66H-GFP which emits less light upon phophorylation. Thereby, the ratio of the two intensities would reveal kinase activity more accurately than only one probe.
Use as real-time probe working at near physiological concentrations. Since the novel proteins are significantly brighter than wild type GFP and prior art derivatives at about 37xc2x0 C. the concentration needed for visualisation can be lowered. Target sites for enzymes engineered into the novel proteins, e.g. F64L-Y66H-GFP or F64L-GFP, can therefore be present in the cell at low concentrations in living cells. This is important for two reasons: 1) The probe must interfere as little as possible with the intracellular process being studied; 2) the translational and transcriptional apparatus should be stressed minimally.
The novel proteins can be used as real time probes based on energy transfer. A probe system based on energy transfer from, e.g. F64L-Y66H-GFP to F64L-GFP.
The novel proteins can be used as reporters to monitor live/dead biomass of organisms, such as fungi. By constitutive expression of F64L-Y66H-GFP or F64L-GFP in fungi the viable biomass will light up.
Transposon vector mutagenesis can be performed using the novel proteins as markers in transcriptional and translational fusions.
Transposons to be used in microorganisms encoding the novel proteins. The transposons may be constructed for translational and transcriptional fusions. To be used for screening for promoters.
Transposon vectors-encoding the novel proteins, such as F64L-Y66H-GFP and F64L-GFP, can be used for tagging plasmids and chromosomes.
Use of the novel proteins enables the study of transfer of conjugative plasmids, since more than one parameter can be followed in living cells. The plasmid may be tagged by F64L-Y66H-GFP or F64L-GFP and the chromosome of the donor/recipient by F64L-Y66H-GFP or F64L-GFP.
Use as a reporter for bacterial detection by introducing the novel proteins into the genome of bacteriophages.
By engineering the novel proteins, e.g. F64L-Y66H-GFP or F64L-GFP, into the genome of a phage a diagnostic tool can be designed. F64L-Y66H-GFP or F64L-GFP will be expressed only upon transfection of the genome into a living host. The host specificity is defined by the bacteriophage.
Any novel feature or combination of features described herein is considered essential to this invention.